Aldehyde conjugated flavonoid preparations

ABSTRACT

There is provided a method of conjugating a polymer containing a free aldehyde group with a flavonoid in the presence of an acid catalyst, such that the polymer is conjugated to the C6 or C8 position of the flavonoid A ring. The resulting conjugates may be used to form delivery vehicles to deliver high doses of flavonoids, and may also be used as delivery vehicles to deliver an additional bioactive agent.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of international applicationPCT/SG2006/000045, filed Mar. 7, 2006, which claims the benefit of U.S.provisional patent application No. 60/682,801, filed on May 20, 2005,the contents of both of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to preparations of flavonoids,and particularly to delivery agent-conjugated flavonoids and/or deliveryagent-conjugated oligomers of flavonoids.

BACKGROUND OF THE INVENTION

Flavonoids are one of the most numerous and best-studied groups of plantpolyphenols. The flavonoids consist of a large group of low-molecularweight polyphenolic substances naturally occurring in fruits andvegetables, and are an integral part of the human diet. Dried green tealeaves can contain as much as 30% flavonoids by weight, including a highpercentage of flavonoids known as catechins (flavan-3-ol derivatives orcatechin-based flavonoids), including (−)-epicatechin,(−)-epigallocatechin, (+)-catechin, (−)-epicatechin gallate and(−)-epigallocatechin gallate.

In recent years, these green tea catechins have attracted much attentionbecause they have been recognized to have biological and pharmacologicalproperties, including antibacterial, antineoplastic, anti-thrombotic,vasodilatory, antioxidant, anti-mutagenic, anti-carcinogenic,hypercholesterolemic, antiviral and anti-inflammatory properties, whichhave been demonstrated in numerous human, animal and in vitro studies(Jankun J., et al. Nature 387, 561 (1997); Bodoni A. et al. J. Nutr.Biochem. 13, 103-111 (2002); Nakagawa K. et al. J. Agric. Food Chem. 47,3967-3973 (1999)). These biological and pharmacological properties arepotentially beneficial in preventing diseases and protecting thestability of the genome. Many of the beneficial effects of catechins arethought to be linked to the antioxidant actions of the catechins (TeraoJ., et al. Arch. Biochem. Biophys. 308, 278-284 (1994)). Among thecatechins, (−)-epigallocatechin gallate (EGCG), which is a majorcomponent of green tea, is thought to have the highest activity,possibly due to the trihydroxy B ring and the gallate ester moiety atthe C3 position (Isemura M., et al. Biofactors 13, 81-85 (2000); IkedaI., et al. J. Nutr. 135, 155 (2005); Lill G., et al. FEBS Letters 546,265-270 (2003); Sakanaka S. and Okada Y. J. Agric. Food Chem. 52,1688-1692 (2004); Yokozawa T., et al., J. Agric. Food Chem. 48,5068-5073 (2000)).

In general, the activity half-life of flavonoids is limited to a fewhours inside the body; metabolism of these compounds has not yet beenestablished. Despite the favorable anti-oxidation and anti-cancerproperties of the catechins including EGCG, it is impractical to achievea therapeutic level of this compound in the body by directly ingesting alarge amount of green tea, due to the inherent volume constraint. Thatis, in order to obtain a therapeutic or pharmacological benefit fromflavonoids through diet alone, it would be necessary to ingest an amountof food and beverage that is larger than is practical to consume.Moreover, pro-oxidant activity has been reported for several flavonoidsincluding EGCG, making ingesting crude green tea directly a lesseffective means of delivering EGCG (Yen G. C., et al. J. Agric. FoodChem. 45, 30-34 (1997); Yamanaka N., et al. FEBS Lett. 401, 230-234(1997); Roedig-Penman A. and Gordon M. H. J. Agric. Food Chem. 1997, 45,4267-4270).

On the other hand, a relatively high-molecular fraction of extractedplant polyphenols (procyanidins) and synthetically oligomerized(+)-catechin and rutin have been reported to exhibit enhancedphysiological properties such as antioxidant and anti-carcinogenicactivity compared to low-molecular weight flavonoids, (Zhao J., et al.Carcinogenesis, 1999, 20, 1737-1745; Ariga T. and Hamano M. Agric. Biol.Chem. 54, 2499-2504 (1990); Chung J. E., et al. Biomacromolecules 5,113-118 (2004); Kurisawa M., et al. Biomacromolecules 4, 1394-1399(2003); Hagerman A. E., et al. J. Agric. Food Chem. 46, 1887 (1998)) andwithout pro-oxidant effects (Hagerman A. E., et al. J. Agric. Food Chem.46, 1887 (1998); Li C. and Xie B. J. Agric. Food Chem. 48, 6362 (2000)).However, neither naturally occurring nor synthesized high molecularweight flavonoids are expected to be absorbed and transported to othertissues after ingestion, since these compounds are typically large, formstrong complexes with proteins and are resistant to degradation (ZhaoJ., et al. Carcinogenesis, 1999, 20, 1737-1745).

In cases of flavonoids consumed via oral intake of foods and beverages,the flavonoids may play a role as antioxidants to protect the digestivetract from oxidative damage during digestion. However, flavonoids can beexpected to remain only in the digestive tract and thus their beneficialphysiological activities are not likely to be utilized to other tissues.Moreover, their strong hydrophobicity as well as their tendency to formcomplexes with proteins makes parenteral delivery of these compoundsdifficult.

Given the beneficial nature of these compounds, it is desirable to findmethods of delivery that would allow for larger quantities to beconsumed, or would provide for the use of catechin-based flavonoids incontexts in which they are not normally found, potentially providingincreased consumption and/or exposure to the catechin-based flavonoids,thereby increasing the potential to receive the pharmacological benefitof these compounds.

SUMMARY OF THE INVENTION

In one aspect, there is provided a conjugate of a delivery agentcontaining a free aldehyde and a flavonoid, having the delivery agentconjugated at the C6 and/or the C8 position of the A ring of theflavonoid.

In another aspect, there is provided a delivery vehicle comprising theconjugate described herein.

In a further aspect, there is provided a method of conjugating adelivery agent having a free aldehyde group in the presence of acid to aflavonoid, comprising reacting the delivery agent with the flavonoid inthe presence of an acid catalyst.

In yet a further aspect, a method of delivering a catechin-basedflavonoid to a subject comprising administering to the subject theconjugate or the delivery vehicle described herein.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate, by way of example only, embodiments ofthe present invention,

FIG. 1A is a schematic depiction of the oligomerization of(−)-epigallaocatechin gallate (EGCG) to yield oligomeric(−)-epigallaocatechin gallate (OEGCG);

FIG. 1B is a schematic depiction of the conjugation of poly(ethyleneglycol) (PEG) and EGCG to yield the PEG-epigallaocatechin gallateconjugate (PEG-EGCG);

FIG. 1C is a schematic depiction of the conjugation of PEG and OEGCG toyield poly(ethylene glycol)-oligomeric epigallaocatechin gallateconjugate (PEG-OEGCG);

FIG. 2A is a schematic depiction of a micellar nanocomplex systemcomprising the self-assembled OEGCG/protein complex surrounded byPEG-EGCG;

FIG. 2B is a schematic depiction of a micellar nanocomplex of aPEG-OEGCG/protein complex;

FIG. 3 is UV-VIS spectra of EGCG, OEGCG, PEG-EGCG, and PEG-OEGCG in anaqueous solution;

FIG. 4 is a DSC thermodiagram for EGCG, OEGCG, PEG-EGCG, PEG-OEGCG, andPEG;

FIG. 5 is plot of ζ-potential of PEG, EGCG, PEG-EGCG, OEGCG, andPEG-OEGCG in PBS;

FIG. 6 is UV-VIS spectra of DPPH solutions treated with EGCG, OEGCG,PEG-EGCG, and PEG-OEGCG, measured at 519 nm;

FIG. 7 is a graph depicting XO inhibition activity of EGCG, OEGCG,PEG-EGCG, PEG-OEGCG, and Allopurinol (n=8);

FIG. 8 is a graph depicting uPA inhibition activity of EGCG, OEGCG,PEG-EGCG, and PEG-OEGCG (n=8);

FIG. 9 is a graph showing the effect of OEGCG and protein concentrationon the size of the micellar nanocomplex;

FIG. 10 is a graph showing the effect on size of the micellarnanocomplex upon PEG-EGCG addition, at varying concentrations of PEG;

FIG. 11 shows the ζ-potential of the various components in PBS;

FIG. 12 is a graph indicating the size of the micellar nanocomplexformed in the presence or absence of OEGCG [a: BSA; b: BSA+PEG; c:BSA+PEG-EGCG; d: (BSA+PEG-EGCG)+BSA; e: PEG-EGCG; f: PEG-EGCG+BSA; g:PEG-EGCG in DMSO; h: (BSA+OEGCG)+PEG-EGCG; i:((BSA+OEGCG)+PEG-EGCG)+BSA];

FIG. 13A is a TEM image of the OEGCG/protein, PEG-EGCG micellarnanocomplex;

FIG. 13B indicates the size distribution of the OEGCG/protein, PEG-EGCGmicellar nanocomplex as measured by light scattering;

FIG. 14 is a graph indicating the size of the complex formed with DNA[∘: OEGCG+DNA; ▪: EGCG+DNA];

FIG. 15 is a graph showing the size of the complex formed in the varioussamples [a: (BSA+OEGCG)+PEG-EGCG; b: OEGCG+PEG-EGCG; c:(OEGCG+PEG-EGCG)+BSA; d: OEGCG+PEG; e: (OEGCG+PEG)+BSA];

FIG. 16 is a graph showing the size of the complex formed in the varioussamples [a: BSA; b: BSA+PEG-OEGCG; c: b after ultrasonication; d:BSA+OEGCG; e: (BSA+OEGCG)+PEG-OEGCG; f: e after ultrasonication];

FIG. 17 is a graph showing the effect of pH on PEG-OEGCG complexation;

FIG. 18 is a graph showing the size of the complex formed in the varioussamples [a: PEG-OEGCG in distilled water; b: EGCG+PEG-OEGCG in distilledwater; c: OEGCG+PEG-OEGCG in distilled water; d: BSA+PEG-OEGCG indistilled water; e: (BSA+EGCG)+PEG-OEGCG in distilled water; f:(BSA+OEGCG)+PEG-OEGCG in distilled water; g: f after replaced in PBS];

FIG. 19 is a representation of the synthesis method of hyaluronicacid-aminoacetylaldehyde diethylacetal conjugate;

FIG. 20 is a representation of the synthesis method of hyaluronicacid-EGCG conjugate;

FIG. 21 is a schematic depiction of synthesis of an hyaluronicacid-tyramine-EGCG (HA-Tyr-EGCG) hydrogel;

FIG. 22A is a graph showing the amount of FITC-BSA released from variousHA-Tyr-EGCG hydrogels;

FIG. 22B is a graph showing the amount of FITC-BSA released from variousHA-Tyr-catechin hydrogels;

FIG. 23A is a graph showing the superoxide scavenging activity ofvarious hyaluronic acid-EGCG (HA-EGCG) conjugates;

FIG. 23B is a graph showing the xanthine oxidase inhibition activity ofvarious HA-EGCG conjugates;

FIG. 24 is a graph showing the urokinase inhibition activity of anHA-EGCG conjugate;

FIG. 25 is a graph showing the size of the BSA/OEGCG complexes by addingurea (triangles), SDS (diamonds), Triton X-100 (squares) and Tween 20(circles);

FIG. 26 is a graph showing the activities of various enzymes upon thecomplexation with OEGCG (white bars) and after the dissociation byTriton X-100 (black bars);

FIG. 27 is a graph showing the ABTS⁺ scavenging activity of variouscomplexes;

FIG. 28 is a graph showing the dose response curves for the inhibitionof various enzymes with increasing weight ratio of OEGCG to protein;

FIG. 29 illustrates the activity recovery of α-amylase with TritonX-100;

FIG. 30 illustrates the activity recovery of lysozyme with Triton X-100;and

FIG. 31 illustrates the activity recovery of xanthine oxidase withTriton X-100.

DETAILED DESCRIPTION

It is generally desirable to find ways to readily increase theconcentration of flavonoids in the body and to improve effectivedelivery of such flavonoids to various tissues in the body.

In order to increase the availability of beneficial flavonoid compounds,the inventors have found that conjugation of flavonoids to variousdelivery agents through a free aldehyde group on the delivery agent tothe A ring of the flavonoid allows for modification of the physicalproperties of the flavonoid without disrupting the polyphenol structureof the flavonoid, while augmenting the biological and pharmacologicalproperties of the flavonoid.

That is, the aldehyde-mediated conjugation of a delivery agent to theflavonoid results in attachment of the delivery agent at the C6 and/orC8 position of the flavonoid A ring, and does not disrupt or affect theB and C rings of the flavonoid or the various hydroxyl groups on theflavonoid.

Conjugation of a delivery agent to a flavonoid can provide a compositionthat is suitable for administration to a subject by incorporating theflavonoid into a particular vehicle formed with the delivery agent, andcan allow for administration of higher concentrations of flavonoids thancan be obtained through diet. The delivery agent can provide stabilityto the composition, resulting in a composition that is metabolized ordegraded more slowly, and which thus may have a longer half-life in thebody than the unconjugated flavonoid alone. For example, the deliveryagent may be of such a nature that the flavonoid is incorporated into acomposition that enhances the water-solubility of the flavonoid, whichcan avoid uptake by the reticuloendothelial system and subsequentclearance by the kidneys, resulting in a longer half-life in the body.Conjugation of other delivery agents may protect the flavonoid fromenzyme degradation.

Thus, there is presently provided a method of conjugating a deliveryagent to a flavonoid comprising reacting the delivery agent with theflavonoid in the presence of an acid catalyst, the delivery agent havinga free aldehyde group, or a group that is able to be converted to a freealdehyde group in the presence of acid.

The flavonoid may be any flavonoid from the general class of moleculesderived from a core phenylbenzyl pyrone structure, and includesflavones, isoflavones, flavonols, flavanones, flavan-3-ols, catechins,anthocyanidins and chalcones. In a particular embodiment the flavonoidis a catechin or a catechin-based flavonoid. A catechin, or acatechin-based flavonoid is any flavonoid that belongs to the classgenerally known as catechins (or flavan-3-ol derivatives), and includescatechin and catechin derivatives, including epicatechin,epigallocatechin, catechin, epicatechin gallate and epigallocatechingallate, and including all possible stereoisomers of catechins orcatechin-based flavonoids. In particular embodiments, the catechin-basedflavonoid is (+)-catechin or (−)-epigallocatechin gallate.(−)-epigallocatechin gallate (EGCG) is thought to have the highestactivity among the catechin-based flavonoids, possibly due to thetrihydroxy B ring and gallate ester moiety at the C3 position of thisflavonoid.

The delivery agent is any chemical group or moiety that contains a freealdehyde or group, or a functional group that can be converted to a freealdehyde group in the presence of acid, for example an acetal group. Thedelivery agent is capable of being formed into a delivery vehicle, thusallowing for the incorporation of a conjugated flavonoid into thedelivery vehicle without compromising the biological or pharmacologicalproperties of the flavonoid. As well, the delivery agent should bebiocompatible, and may be biodegradable in some embodiments.

The following discussion refers to an embodiment in which the flavonoidis a catechin-based flavonoid and in which the delivery agent is apolymer. However, it will be understood that the aldehyde condensationreaction between an aldehyde-containing chemical group and a flavonoidis applicable to conjugation of any delivery agent having a freealdehyde group, including following acid treatment of the deliveryagent, to any flavonoid, as described above.

Thus, in one embodiment the method involves conjugation of a polymercontaining a free aldehyde group or a group that is able to be convertedto a free aldehyde group in the presence of acid to a catechin-basedflavonoid.

The catechin-based flavonoid may be a single monomeric unit of acatechin-based flavonoid or it may be an oligomer of one or morecatechin-based flavonoids. As stated above, conjugation of a polymer toa flavonoid results in augmentation of the flavonoid's biological orpharmacological properties. As well, an oligomer of the catechin-basedflavonoid tends to have amplified or augmented levels of the biologicaland pharmacological properties associated with catechin-basedflavonoids, and may even have reduced pro-oxidant effects that aresometimes associated with monomeric catechin-based flavonoids. Thus, inone embodiment, the catechin-based flavonoid is an oligomerizedcatechin-based flavonoid having amplified or augmented flavonoidproperties.

Oligomers of catechin-based flavonoids are known, including oligomersprepared through enzyme-catalyzed oxidative coupling and throughaldehyde-mediated oligomerization. An aldehyde-mediated oligomerizationprocess results in an unbranched oligomer that has defined linkages, forexample through carbon-carbon linkages such as CH—CH₃ bridges linkedfrom the C6 or C8 position on the A ring of one monomer to the C6 or C8position on the A ring of the next monomer, including in either possiblestereoconfiguration, where applicable. Thus, the CH—CH₃ linkage maybetween the C6 position of the A ring of one monomer and either of theC6 or C8 position of the next monomer or it may be between the C8position of the A ring of the first monomer and either of the C6 or C8position of the next monomer. For example, FIG. 1A depicts oligomeric(−)-epigallocatechin gallate (OEGCG) produced from an aldehyde-mediatedoligomerization method, which is connected through C6-C8 linkages of(−)-epigallocatechin gallate monomers.

The oligomer of the catechin-based flavonoid may be of 2 or moremonomeric units linked together. In certain embodiments, thecatechin-based flavonoid oligomer has from 2 to 100 flavonoid monomerunits, from 10 to 100, from 2 to 80, from 10 to 80, from 2 to 50, from10 to 50, from 2 to 30, from 10 to 30, from 20 to 100, from 30 to 100 orfrom 50 to 100 monomeric units.

The polymer may be any polymer having a free aldehyde group prior toconjugation with the catechin-based flavonoid, or having a group that isconverted to an aldehyde group in the presence of acid, for example anacetal group. Furthermore, it will be understood that the polymer shouldbe non-toxic, biocompatible and suitable for pharmacological use. Thepolymer may also have other desirable properties, for example, thepolymer may have low immunogenicity, and it may be biodegradable ornon-biodegradable depending on the desired biological application of thecomposition, for example, for controlled release of catechin-basedflavonoids or other bioactive agents at a particular site in a body.

The polymer may be chosen based on its particular characteristics andits ability to form certain types of delivery vehicles. For example, thepolymer may be an aldehyde-terminated poly(ethylene glycol), or it maybe hyaluronic acid derivatized with an aldehyde group, or a derivativeof such polymers. Alternatively, the polymer may be aphenoxymethyl(methylhydrazono) dendrimer (PMMH), for example,cyclotriphosphazene core PMMH or thiophosphoryl core PMMH. The polymermay also be any biological polymer, modified to contain a free aldehydegroup or a group that is convertible to an aldehyde in the presence ofacid, for example an aldehyde-modified protein, peptide, polysaccharideor nucleic acid. In one particular embodiment the polymer is analdehyde-terminated poly(ethylene glycol) (PEG-CHO). In anotherparticular embodiment, the polymer is aldehyde-derivatized hyaluronicacid, hyaluronic acid conjugated with aminoacetylaldehyde diethylacetal,or either of the aforementioned hyaluronic acid polymers derivatizedwith tyramine.

The free aldehyde group on the polymer allows for the conjugation of thepolymer in a controlled manner to either the C6 or the C8 position ofthe A ring, or both, of the flavonoid structure, thus preventingdisruption of the flavonoid structure, particularly the B and C rings ofthe flavonoid, and thus preserving the beneficial biological andpharmacological properties of the flavonoid.

The polymer is conjugated to the catechin-based flavonoid via a reactionof the aldehyde group of the polymer with the C6 and/or the C8 positionof the A ring of the catechin-based flavonoid, as shown in FIG. 1B andFIG. 1C.

The conjugate is synthesized using acid catalysis of a condensation ofthe aldehyde group of the polymer with the catechin-based flavonoid, orusing acid to convert a functional group on the polymer to a freealdehyde prior to condensation of the aldehyde group with thecatechin-based flavonoid.

To conjugate the polymer and the catechin-based flavonoid, the polymerand the catechin-based flavonoid may be separately dissolved in asuitable solvent. The polymer with the free aldehyde is added, forexample by dropwise addition, to the solution containing thecatechin-based flavonoid, in the presence of an acid. The reaction isallowed to go to completion. Following the conjugation reaction, excessunreacted polymer or catechin-based flavonoid can be removed from theconjugated composition, for example by dialysis or by molecular sieving.

The ratio of catechin-based flavonoid to polymer may be varied, so thatthere is only one polymer moiety attached to the catechin-basedflavonoid portion of the polymer, or so that there is a catechin-basedflavonoid portion attached at more than one position on the polymer, orso that the catechin-based flavonoid portion has two polymer portionsattached, one at either of the C6 and C8 positions of the catechin-basedflavonoid.

The ratio of polymer to catechin-based flavonoid in the finalcomposition can be controlled through the ratio of starting reagents.For example, when the molar ratio of polymer moiety to catechin-basedflavonoid moiety is about 1, a single polymer moiety will be attached toa single catechin-based flavonoid moiety (either monomeric or oligomericmay be used). However, at higher concentrations of polymer, for exampleat a 10:1 molar ratio of polymer to catechin-based flavonoid, acomposition having a tri-block structure of polymer-flavonoid-polymermay be obtained.

A conjugate of a polymer containing a free aldehyde and a catechin-basedflavonoid, having the polymer conjugated at the C6 and/or the C8position of the A ring of the flavonoid is also contemplated.

Conjugation of the polymer also allows for the incorporation ofcatechin-based flavonoids into various compositions or vehicles. Byselection of the particular polymer containing a free aldehyde groupbased on the physical properties of the polymer, it is possible toincorporate flavonoids into a variety of different vehicle types,allowing for the delivery of high concentrations of flavonoids indifferent contexts to various targeted areas of the body.

Thus, the present conjugate resulting from the above-described methodmay be formed into a delivery vehicle, depending on the nature of thepolymer portion of the conjugate. The delivery vehicle may be used todeliver the catechin-based flavonoid to a body, including a particulartargeted site in a body, depending on the nature of the deliveryvehicle. Optionally, a bioactive agent may be included in the deliveryvehicle, which may then be simultaneously delivered to the site in thebody. Thus, there is provided a delivery vehicle comprising acomposition that comprises a catechin-based flavonoid conjugated to apolymer through a free aldehyde group on the polymer, the deliveryvehicle optionally further comprising a bioactive agent.

The bioactive agent may be any agent that has a biological,pharmacological or therapeutic effect in a body, and includes a protein,a nucleic acid, a small molecule or a drug. A bioactive agent that is aprotein may be a peptide, an antibody, a hormone, an enzyme, a growthfactor, or a cytokine. A bioactive agent that is a nucleic acid may besingle stranded or double stranded DNA or RNA, a short hairpin RNA, ansiRNA, or may comprise a gene encoding a therapeutic product. Alsoincluded in the scope of bioactive agent are antibiotics,chemotherapeutic agents and antihypertensive agents.

In one particular embodiment, the delivery vehicle is a micellarnanocomplex, which is suitable for parenteral delivery of catechin-basedflavonoids, and optionally bioactive agents to a particular site withina body. The polymer is chosen to have properties that allow it toassemble with the catechin-based flavonoid portion of the composition,protecting the flavonoid from the solution environment. If a suitablesolvent is chosen in which the polymer portion of the conjugate issoluble and is more soluble than the catechin-based flavonoid, theconjugate will self-assemble, excluding the solution from the flavonoidcore, thus allowing for assembly of micellar complexes.

In a particular embodiment of the micellar nanocomplex delivery vehicle,the polymer chosen is aldehyde-terminated PEG, or a derivative thereof.PEG is a polymer widely used as a pharmacological ingredient, andpossesses good hydrophilic, non-toxic, non-immunogenic andbiocompatibility characteristics with low biodegradability.

By conjugating PEG-CHO to a catechin-based flavonoid, a conjugate isformed that has strong self-assembly tendencies. In one embodiment, PEGis conjugated to a monomer of a catechin-based flavonoid, to form aPEG-flavonoid. The delivery vehicle is formed together withnon-conjugated catechin-based flavonoids, and optionally a bioactiveagent. Thus, the central core contains relatively high concentrations ofa flavonoid and the external shell of the micellar nanocomplex comprisesthe conjugated PEG-monomeric flavonoid, and is assembled in a two-stepprocess. In a particular embodiment, the central core is oligomeric EGCGand the external core is made up of conjugated PEG-EGCG.

Formation of this two-step assembly of the delivery vehicle results intemporary partial or complete masking of the biological activities ofthe oligomeric flavonoid that is incorporated into the core of thedelivery vehicle. For example, while assembled into core of the deliveryvehicle, the augmented properties of the oligomerized EGCG are lessavailable, due to physical interactions with other molecules in theassembled core portion of the delivery vehicle. Upon release from thedelivery vehicle, for example by fusion of the vehicle with a cellularphospholipid membrane, the components of the delivery dissociate,unmasking the biological properties of the oligomeric catechin-basedflavonoid.

This embodiment of the delivery vehicle is well suited to deliverbioactive agents. Since the catechin-based flavonoids have a rigid,multi-ring core structure, these molecules associate well with bioactiveagents such as proteins and nucleic acids, as well as other moleculescontaining ring structures, likely by stacking of the catechin ringswith the ring or rings on the bioactive agent. Thus, an oligomericcatechin-based flavonoid can be used to associate with the bioactiveagent prior to assembly in the micellar nanocomplex, as shown in FIG.2A.

The concentration of the bioactive agent is chosen depending on thetotal amount of bioactive agent that is to be delivered to a particularsite in a body, and on the amount of bioactive agent that can beincluded in the micellar nanocomplex without destabilizing the micellarstructure. In certain embodiments, up to 50%, or up to 40%, w/w of themicellar complex may comprise the bioactive agent.

The biological activity of the bioactive agent is also temporarilypartially or completely masked while incorporated into the presentdelivery vehicles. As with the oligomeric catechin-based flavonoid, thebiological properties of the bioactive agent are masked or sequestered,making them less available while the bioactive agent is assembled in thedelivery vehicle, meaning that the bioactive agent is not able to exertbioactivity or interact with other molecules in a bioactive manner whilecontained in the delivery vehicle. Upon release of the bioactive agentfrom the delivery vehicle, the biological properties of the bioactiveagent are once again available, and the bioactive agent is able to exerta biological effect.

In another embodiment, PEG is conjugated to an oligomeric catechin-basedflavonoid. This embodiment of the delivery agent has strongself-assembly properties and can be self-assembled in a single stepprocess. As with the two-step assembly micellar nanocomplex above, thesingle-step assembling micellar nanocomplex may optionally include abioactive agent. FIG. 2B describes a nanocomplex comprising PEG-OEGCGand a protein.

The above micellar nanocomplexes are of nanoscale dimensions, and may befrom about 1 nm to about 10000 nm in diameter, or from about 20 nm toabout 4000 nm in diameter, or from about 20 nm to about 100 nm indiameter. The size of the micellar nanocomplexes can be varied byvarying the length of the oligomerized catechin-based flavonoid, thelength of the polymer, and the concentration of unconjugatedoligomerized catechin-based flavonoid. The size of the micellarnanocomplex may be pH dependent, depending on the polymer used. Forexample, in micellar nanocomplexes in which the conjugated polymer isPEG, the diameter of the micelles tends to decrease with increasing pH.

Generally, the micellar nanocomplexes undergo self assembly and thuslittle synthesis is required. For the two step process, the componentsthat are to form the core are dissolved in a suitable solvent, forexample in diluted DMSO or methanol, and are allowed to assemble. Thesolvent is a solvent in which the core components are soluble, and whichmay be miscible in water, or which may be volatile, or from which theassembled micelles can otherwise be isolated or extracted. As indicatedabove, the core components may be for example a bioactive agent and acatechin-based flavonoid, for example an oligomeric catechin-basedflavonoid. The polymer-catechin-based flavonoid conjugate that is toform the outer shell is then added to the solution and the micellarcomplex is allowed to form.

For the one step self-assembly process, the polymer-catechin-basedflavonoid conjugate, optionally with a bioactive agent, is dissolved ina suitable buffer as described for the two-step process and the micellarnanocomplex is allowed to assemble.

This micellar nanocomplex system provides the ability to achievecontrolled biodistribution of catechin-based flavonoids and prolongedcirculation half-life in bloodstream due to the PEG outer shell, as wellas amplified pathological activities of the catechin-based flavonoidcompound, with the added benefit that such compounds may be accompaniedby therapeutic effect of an additional bioactive agent loaded in theinner core of the micelle. Where the bioactive agent is a sensitivemolecule such as a protein, the nanoscale micelles offer a convenientdelivery vehicle with the advantage of a gentle, self-assembly methodthat does not involve the mechanical, thermal and chemical stresses thatcan be associated with conventional encapsulation techniques currentlyused, which conventional techniques may lead to denaturation ofsensitive bioactive agents such as proteins.

In another particular embodiment, the delivery vehicle is a hydrogel,which can be used as a wound or burn dressing, for sustained releasedelivery of a bioactive agent, as a support for tissue regeneration, fortreatment of arthritis, or for cosmetic applications such as a facialmask.

The polymer is chosen to have good swellability characteristics and tohave appropriate groups available for cross-linking of the polymermoieties, and to be non-toxic and biocompatible, and in some embodimentsto be biodegradable.

In a particular embodiment of the hydrogel, the polymer is aldehydederivatized hyaluronic acid, or a derivative of hyaluronic acid such ashyaluronic acid aminoacetylaldehyde diethylacetal conjugate, or atyramine derivative of aldehyde-derivatized hyaluronic acid orhyaluronic acid aminoacetylaldehyde diethylacetal conjugate.

Conjugates comprising a hyaluronic acid-catechin-based flavonoid can bereadily cross-linked to form a hydrogel, without disruption of thebiological or pharmacological properties of the flavonoid. Suchhydrogels may also optionally comprise a bioactive agent as describedabove, for release of the bioactive agent at the site where the hydrogelis applied.

The hyaluronic acid-flavonoid conjugate is synthesized by reacting thehyaluronic acid with the catechin-based flavonoid under acidicconditions, for example from about 1 to about 5, or for example at pH ofabout 1. The conjugated polymer-flavonoid is then purified, for exampleby dialysis, and then mixed with bioactive agent and a cross-linkingagent, such as hydrogen peroxide. A cross-linking catalyst is added, forexample horseradish peroxidase, and the hydrogel may then be quicklypoured in to a mold to form a desired shape before the cross-linkingreaction is completed. For example, the hydrogel may be formed into aslab suitable for application as a wound dressing.

The components of the hydrogel may also be injected and reacted to formthe hydrogel in vivo, for example by injecting an uncrosslinkedconjugate, optionally with a bioactive agent, together with across-linking agent, such as hydrogen peroxide and a cross-linkingcatalyst, for example, horseradish peroxidase. Such a hydrogel is usefulfor drug delivery to a specific site in a body, or for tissueengineering.

Since hyaluronic acid has multiple sites that may react with theflavonoid during the conjugation reaction, by varying the concentrationof the catechin-based flavonoid in the starting reaction, it is possibleto vary the degree of conjugation between the hyaluronic acid polymerand the catechin-based flavonoid. For example, the ratio of reactantsmay be adjusted so that the resulting conjugate has from about 1% toabout 10% of the sites on the polymer conjugated with the flavonoid.Alternatively, additional hyaluronic acid that has not been conjugatedcan be added to the mixture prior to cross-linking of the hydrogel sothat some of the polymer molecules in the hydrogel will not beconjugated to the flavonoid.

The above described compositions and delivery vehicles are well-suitedfor controlled and targeted delivery of catechin-based flavonoids toparticular sites within the body. The flavonoids can provideantibacterial, antineoplastic, anti-thrombotic, vasodilatory,antioxidant, anti-mutagenic, anti-carcinogenic, hypercholesterolemic,antiviral and anti-inflammatory activity at the targeted site. Thus, theabove conjugates and delivery vehicles are useful for a variety oftreatment applications. In addition, the delivery vehicles can includean additional bioactive agent, making the delivery vehicles useful inthe treatment of a wide range of disorders or diseases. For example,immunoregulatory peptides and proteins including cytokines and growthfactors have emerged as an important class of drugs for the treatment ofcancer, myelodepresssion and infectious disease.

Thus, there is presently provided a method of delivering acatechin-based flavonoid to a subject comprising administering aconjugate of a polymer containing a free aldehyde and a catechin-basedflavonoid, having the polymer conjugated at the C6 and/or the C8position of the A ring of the flavonoid is also contemplated, asdescribed above. In certain embodiments, the conjugate is formed into adelivery vehicle, such as a micellar nanocomplex or a hydrogel, asdescribed above.

The subject is any animal, including a human, in need of catechin-basedflavonoids, and may be in further need of an additional bioactive agent.

The conjugate may be administered using known methods, which will dependon the form of the conjugate. Non-oral routes are preferred,particularly if a bioactive agent is being administered simultaneouslyin the same form with the conjugate. If the conjugate is formulated as asolution, or in the form of micellar nanoparticles, the conjugate may bedelivered parenterally, including intravenously, intramuscularly, or bydirect injection into a targeted tissue or organ. If the conjugate isformulated as a hydrogel, the conjugate may be applied topically or bysurgical insertion at a wound site.

The conjugate may be administered in combination with a bioactive agent,particularly where the conjugate is formulated as a delivery vehicle asdescribed above.

When administered to a patient, the conjugate is administered in anamount effective and at the dosages and for sufficient time period toachieve a desired result. For example, the conjugate may be administeredin quantities and dosages necessary to deliver a catechin-basedflavonoid which may function to alleviate, improve, mitigate,ameliorate, stabilize, prevent the spread of, slow or delay theprogression of or cure an infection, disease or disorder, or to inhibit,reduce or impair the activity of a disease-related enzyme. Adisease-related enzyme is an enzyme involved in a metabolic orbiochemical pathway, which when the pathway is interrupted, or whenregulatory control of the enzyme or pathway is interrupted or inhibited,the activity of the enzyme is involved in the onset or progression of adisease or disorder.

The effective amount of conjugate to be administered to a subject canvary depending on many factors such as the pharmacodynamic properties ofthe conjugate, including the polymer moiety and the catechin-basedflavonoid moiety, the mode of administration, the age, health and weightof the subject, the nature and extent of the disorder or disease state,the frequency of the treatment and the type of concurrent treatment, ifany, and the concentration and form of the conjugate.

One of skill in the art can determine the appropriate amount based onthe above factors. The conjugate may be administered initially in asuitable amount that may be adjusted as required, depending on theclinical response of the subject. The effective amount of conjugate canbe determined empirically and depends on the maximal amount of theconjugate that can be administered safely. However, the amount ofconjugate administered should be the minimal amount that produces thedesired result.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES

(−)-Epigallocatechin gallate (EGCG), a main ingredient of green tea,exhibits numerous biological and pharmacological effects. In thefollowing examples, conjugates of poly(ethylene glycol) with EGCG oroligomeric EGCG (OEGCG) were synthesized using aldehyde-mediatedcondensation by an acid catalyst. The synthesized compounds werecharacterized with molecular weight, NMR spectra, phenolic analysis,UV-VIS spectra, DSC thermogram, and ζ-potential.

Example 1 Conjugation of Polyethylene Glycol with (−)-epigallocatechinGallate or with Oligomeric (−)-epigallocatechin Gallate

In this study, we synthesized conjugates of poly(ethylene glycol) (PEG)with (−)-epigallocatechin gallate (EGCG) or oligomerized EGCG (OEGCG).PEG-EGCG or PEG-OEGCG conjugation using aldehyde-terminated PEG(PEG-CHO) was carried out by Baeyer acid-catalyzed condensation betweenan aldehyde moiety at the end of PEG chain and a nucleophilicphloroglucinol ring of the EGCG moiety (FIG. 1).

Materials: (−)-Epigallocatechin gallate (EGCG) was purchased from KURITALTD., Japan. Aldehyde-terminated polyethylene glycol (PEG-CHO) waspurchased from NOF Co., Japan. Acetic acid, acetaldehyde, PURPALD™,Folin-Ciocalteau phenol reagent, sodium carbonate, vanillin anddimethylsulfoxide-d₆ were purchased from Sigma-Aldrich. 1N sodiumhydroxide was purchased from Wako Pure Chemical Industries, Japan. Otherreagents and solvents are commercially available and used as received.

Synthesis of PEG-epigallocatechin gallate conjugate: PEG-CHO and EGCGwere separately dissolved in a mixture of acetic acid/water/ethanol oracetic acid/water/DMSO. The molar ratio of EGCG was varied in excess toPEG-CHO. The reaction was started by dropwise addition of PEG-CHOsolution and performed at 20° C. (pH from 1 to 5) under air or nitrogenatmosphere for varied reaction time. The resulting products weredialyzed (molecular weight cutoff: 3.5×10³) against 1000 times thevolume of methanol at room temperature for two days. The dialysate wasreplaced to distilled water six times and the remaining solution waslyophilized to give the conjugates of PEG and (−)-epigallocatechingallate, or PEG-EGCG.

¹H NMR (DMSO-d₆): δ 2.6-3.0 (H-4 of C ring), 3.2-3.7 (CH₃O and CH₂CH₂Oof PEG), 4.9-5.0 (H-2 of C ring), 5.5 (H-3 of C ring), 5.8-6.0 (H-6 and8 of A ring), 6.3-6.5 (H-2″ and 6″ of galloyl moiety), 6.7-6.9 (H-2′ and6′ of B ring).

¹³C NMR (DMSO-d₆): δ 31.5 (C-4 of C ring), 47.8-49 (CH₂CHO of PEG), 58.9(CH₃O of PEG), 70.7-72.1 (CH₂CH₂O of PEG), 106.3-106.4 (C-2′ and 6′ of Bring), 109.5 (C-2″ and 6″ of galloyl moiety), 146.2-146.4 (C-3′ and 5′of B ring and C-3″ and 5″ of galloyl moiety).

Synthesis of oligomeric epigallocatechin gallate: EGCG was dissolved ina mixture of acetic acid/water/DMSO or acetic acid/water/ethanol. Thereaction was started by addition of acetaldehyde and performed at 20° C.(pH from 1 to 5) under air or a nitrogen atmosphere for varied reactiontime. The resulting products were dialyzed (molecular weight cutoff:1×10³) in a same way described above. The remaining solution waslyophilized to give oligomeric epigallocatechin gallate (OEGCG).

¹H NMR (DMSO-d₆): δ 1.1-1.9 (CHCH₃), 2.6-3.1 (H-4 of C ring), 3.0-3.5(H-3 of C ring), 4.9-5.1 (H-2 of C ring), 5.1-5.4 (CHCH₃), 6.4-6.5 (H-2″and 6″ of galloyl moiety), 6.8-6.9 (H-2′ and 6′ of B ring).

¹³C NMR (DMSO-d₆): δ 15.6-19 (CHCH₃), 19-24 (CHCH₃), 26.6-27.4 (C-4 of Cring), 68.5-68.6 (C-3 of C-ring), 77.3-77.4 (C-2 of C ring), 106.2-106.3(C-2′ and 6′ of B ring), 109.5-109.6 (C-2″ and 6″ of galloyl moiety),120.0-120.1 (C-1″ of galloyl moiety), 129.3-129.5 (C-4c of A ring),133.1-133.2 (C-1′ of B ring), 139.4 (C-4′ of B ring and C-4″ of galloylmoiety), 146.2-146.5 (C-3′ and 5′ of B ring and C-3″ and 5″ of galloylmoiety), 150-158 (C-5, 7 and 8b of A ring), 166 (C-a of galloyl moiety).

Synthesis of PEG-oligomeric epigallocatechin gallate: PEG-CHO wasdissolved in a mixture of acetic acid/water/ethanol or aceticacid/water/DMSO. OEGCG was dissolved in a same solvent with variousmolar ratios (0.1-1) to those of PEG-CHO. The solution of PEG-CHO wasadded dropwise and the reaction was carried out at 20-50° C. (pH from 1to 5) under air or nitrogen atmosphere for varied reaction time. Theresulting opaque products were dialyzed (molecular weight cutoff: 5000)in a same way described above. After centrifugation (rpm=3.5×10⁴) theprecipitate was collected and washed by distilled water in triplicate,followed by lyophilization to give the conjugate of PEG and oligomeric(−)-epigallocatechin gallate (PEG-OEGCG).

¹H NMR (DMSO-d₆): δ 1.1-1.5 (CHCH₃), 2.6-3.1 (H-4 of C ring), 3.2-3.7(CH₃O and CH₂CH₂O of PEG), 4.9-5.0 (H-2 of C ring), 5.1-5.4 (CHCH₃),6.4-6.5 (H-2″ and 6″ of galloyl moiety), 6.8-6.9 (H-2′ and 6′ of Bring).

¹³C NMR (DMSO-d₆): δ 70.7-72.2 (CH₂CH₂O of PEG), 77.3-77.4 (C-2 of Cring), 106.2-106.4 (C-2′ and 6′ of B ring), 109.5-109.6 (C-2″ and 6″ ofgalloyl moiety), 120.0-120.1 (C-1″ of galloyl moiety), 129.3-129.5 (C-4cof A ring), 133.1-133.2 (C-1′ of B ring), 139.4 (C-4′ of B ring and C-4″of galloyl moiety), 146.2-146.5 (C-3′ and 5′ of B ring and C-3″ and 5″of galloyl moiety), 150-158 (C-5, 7 and 8b of A ring), 166 (C-a ofgalloyl moiety).

Measurements: Molecular weight was estimated by size exclusionchromatography (SEC) (Waters 2690 equipped with RI-2410 detector,polystyrene standard) with Waters Styragel HR4E/HR5E columns using THFas an eluant at a flow rate of 1 ml/min at 40° C., after acetylation. ¹Hand ¹³C NMR were recorded on a Bruker 400-MHz nuclear magnetic resonance(NMR) spectrometer.

The aldehyde moiety of unreacted PEG-CHO was quantitatively assessedusing 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (PURPALD™) which isexceedingly specific and sensitive to aldehydes and which yieldspurple-to-magenta-colored 6-mercatriazolo-[4,3-b]-s-tetrazines.^(31,32)100 μl of a sample solution was dropped into 3 ml of the PURPALD™solution (7.5 mg/ml 1N NaOH). After aeration at room temperature, theabsorption maxima of the solutions were recorded at 545 nm using UV-VISspectrometer (JASCO V-510 UV/VIS/NIR spectrometer, Japan). Since thePURPALD™ is sensitive to even a small amount of aldehyde present in air,and resultingly produces a color reaction, a negative control in air wasmeasured and subtracted from the values. Unreacted PEG-CHO wasdetermined using PEG-CHO standard curve. The results of this PURPALD™assay were compared to that of NMR measurement.

Phenolic content of conjugates was assessed by Folin-Ciocalteu assay andvanillin-HCl assay. Folin-Ciocalteu assay has been used for totalphenolics determination by many researchers (Julkunen-tiitto R. J.Agric. Food Chem. 33, 21-217 (1985)). 15 μl of sample was added to 300μl of water. 150 μl of Folin-Ciocalteu phenol reagent was added and thesolution was vigorously shaken. Immediately, 750 μl of 20% sodiumcarbonate solution was added and the mixture was made up to 1.5 ml withwater, following shaking again. After 20 min the absorptivity of themixture was read at 720 nm using a UV-VIS spectrometer. Vanillin-HClmethod has been used for catechins and condensed tannin determination(Broadhurst R. B. and Jones W. T. J. Sci. Food Afric. 29, 788-794(1978)). For this assay, 100 μl of a sample was added to 1 ml of 4%vanillin in methanol and the mixture was shaken vigorously. 0.5 ml ofconcentrated HCl was added then, and the mixture was immediately shakenagain. The absorptivity was read at 500 nm after keeping the mixture atroom temperature for 20 min. The phenolic content of synthesizedcompounds using these two assays was determined using EGCG standardcurves measured in the same manners. Each measurement was run intriplicate.

The melting temperatures (T_(m)) of products were measured with DSCQ100TA Instruments. The measurements were calibrated using indium andcarried out at temperatures from −40 to 200° C. under nitrogen purge ata scanning rate of 20° C./min. ζ-potential of sample solutions wasdetermined by ZetaPALS Zeta Potential Analyzer (BROOKHAVEN INSTRUMENTSCo.) at 25° C. Each measurement was run in triplicate.

Aldehyde-mediated conjugation between polyethylene glycol and(−)-epigallocatechin gallate or oligomerized (−)-epigallocatechingallate: In this study, conjugation of polyethylene glycol (PEG) withEGCG or oligomeric EGCG (OEGCG) was carried out using analdehyde-mediated condensation by an acid catalyst. Synthesis of OEGCGis summarized in Table 1. The reaction was carried out with an excess ofacetaldehyde under varied reaction conditions. Oligomers were obtainedwith several thousands molecular weight after purification by dialysis(MWCO=1000). The molecular weight was measured by SEC after acetylationsince interaction between the many hydroxyl groups present on the EGCGunits and the SEC column results in a lower estimation of molecularweight. Both molecular weight and yields were not affected by reactiontime but were very affected by solvents and reaction atmosphere: boththe molecular weight and yields were higher in the dimethylsulfoxide(DMSO) and water mixture than the ethanol and water mixture, although anincrease in the amount of water in the solvent mixture decreasedmolecular weight and yield. The reaction in N₂ atmosphere producedhigher molecular weight and yields, may be due to O₂ in air terminatingthe oligomerization of EGCG. Resulting oligomers were also soluble ingood solvents for EGCG, such as DMSO, N,N-dimethylformamide, acetone,ethanol, methanol, tetrahydrofuran and an alkaline aqueous solutionexcept for water, and not soluble in chloroform and hexane in whichneither was EGCG.

¹H and ¹³C NMR analysis of the product revealed that condensation ofEGCG in the presence of acetaldehyde gave EGCG oligomers linked througha CH—CH₃ bridge at the C6 and C8 position of the phloroglucinol ring (Aring) (FIG. 1). Singlet peaks due to H6 and H8 of A ring observed at δ¹H 5.83 and 5.93 disappeared after oligomerization, and new peaks due tothe methyl and methine protons of the CH—CH₃ bridge appeared at δ ¹H1.48 and 5.08 (δ ¹³C 21.2 and 16.2), respectively. All peaks for OEGCGwere broadened and have lower intensity compared with those for EGCG.

Conjugation of PEG with EGCG and OEGCG was summarized in Table 2 andTable 3, respectively. After PEG-EGCG conjugation was carried out,unreacted EGCG was removed by dialysis (MWCO=3.5×10³). In order tocompletely consume PEG-CHO, an excess amount of EGCG was fed into thereactor. When EGCG was fed with a 20 times larger molar amount than thatof PEG-CHO in a N₂ atmosphere, the product was shown to contain nounreacted PEG-CHO, as analyzed by NMR (δ 9.65 (s, CHO)) andspectrophotometric assay using PURPALD™.

The molecular weight of conjugates showed that only one chain of PEG-CHOwas conjugated to the EGCG, even though EGCG has two available linkpositions for aldehyde at C6 and C8 (Table 2). This may be due to sterichindrance following the conjugation of a single PEG chain at either ofthe C6 and C8 positions of A ring. However, PEG-OEGCG conjugates wereobtained as both bi- and tri-block conjugates (PEG-OEGCG andPEG-OEGCG-PEG), when PEG-CHO was fed with a ten times lager molar amountthan that of OEGCG (Table 3). By feeding with same molar ratio of OEGCGand PEG-CHO, the conjugation produced bi-block conjugate alone withouttri-block conjugate.

The reaction in DMSO and a N₂ atmosphere resulted in high yields, as inthe case of EGCG oligomerization mentioned above. All PEG-OEGCGconjugates were not water soluble including a conjugate of longer chainPEG with Mn=10000, while all of PEG-EGCG conjugates were water soluble.PEG-OEGCG was separated by centrifugation of an opaque aqueous solutionafter unreacted OEGCG was removed by dialysis against methanol. NMRanalysis revealed that the supernatant was unreacted PEG-CHO and theprecipitate was PEG-OEGCG conjugates. ¹H and ¹³C NMR spectra of PEG-EGCGconjugate exhibited all intrinsic peaks belonged to PEG and EGCG, andthe spectra of PEG-OEGCG also showed broadened peaks for OEGCG includingCHCH₃ bridges as well as for PEG (FIG. 1).

Phenolic determination of conjugates: The EGCG moiety content ofPEG-EGCG and PEG-OEGCG conjugates was assessed using vanillin andFolin-Ciocalteau assays which are commonly used for phenolicquantification in plant material. Folin-Ciocalteau assay is a proteindetermination method used for detecting tyrosine, tryptophan andcysteine residue of proteins (Folin O. and Ciocalteu U. J. Biol. Chem.73, 62-650 (1927)). This assay is nonspecific for phenol groups and alsoreacts with urea, chitosan and guanine to yield deep blue compounds.Vanillin-concentrated HCl assay (Broadhurst R. B. and Jones W. T. J.Sci. Food Afric. 29, 788-794 (1978)) is frequently used to detectcatechins and procyanidins (condensed tannin). Standard curves wereprepared using EGCG. All the standards tested in both assays showed alinear relationship between absorptivity and standard concentrationvarying in a range from 125 μM to 4 mM and from 62.5 μM to 2 mM forFolin-Ciocalteau and vanillin assay, respectively. The vanillin assayfor PEG-EGCG and PEG-OEGCG solutions was quite reproducible and gavenearly same amount as the concentrations that were calculated based ontheir molecular weight. However, the Folin-Ciocalteau assay yielded48.3±18.9% and 126.5±43.2% higher concentrations than the concentrationscalculated based on molecular weight for PEG-EGCG and PEG-OEGCG,respectively.

Optical property: FIG. 3 depicts the UV-VIS spectra of OEGCG, PEG-EGCGand PEG-OEGCG. These compounds were characterized in a manner similar tothat of the precursors. EGCG showed an absorption maximum at 280 nm,indicating that the original flavanic skeleton is retained. In addition,we found that polymerized (+)-catechin by oxidative coupling usingenzyme catalysts showed another absorption maxima at 388 nm in additionto that at 280 nm, giving a complicated structure, while (+)-catechincondensed through a CH—CH₃ bridge showed absorption maximum only at 280nm. Therefore, the UV-VIS spectra of the present OEGCG, PEG-EGCG andPEG-OEGCG were considered further evidence for their structure shown byNMR as described above.

Thermal property: Thermal property of OEGCG, PEG-EGCG and PEGC-OEGCG wascharacterized by DSC measurement (FIG. 4). Endotherm peaks whichcorrespond to the melting point (T_(m)) of PEG and EGCG were observed at62.0 and 150.5° C., respectively. OEGCG showed a broadened Tm peakshifted to lower temperature compared to EGCG, reflecting a decrease incrystallinity. The DSC thermograms of PEG-EGCG and PEG-OEGCG had bimodalpeaks corresponding to PEG and EGCG or OEGCG. T_(m)s originating fromPEG were also shifted to lower temperature and the heat capacity of PEGin melting (ΔH_(Tm)) became smaller, with a 26% and 73% decrease whenconjugated with EGCG and OEGCG, respectively. These data indicate thatthe conjugation and oligomerization occurred as described.

ζ-potential: ζ-potential of the oligomers and conjugates were measuredin PBS (FIG. 5). PEG and EGCG exhibited a slightly negative surfacecharge having similar values, and the PEG-EGCG conjugate showed nodifference in charge compared to EGCG. On the other hand, OEGCG revealeda more negative charge than that of EGCG and the conjugation of OEGCGwith PEG resulted in an apparently stronger negative charge than bothPEG-EGCG and OEGCG.

Example 2 Augmentation of Physiological Activity of (−)-epigallocatechinGallate by Oligomerization and Conjugation with PEG

Materials: (−)-Epigallocatechin gallate (EGCG) was purchased from KURITALTD., Japan. Aldehyde-terminated polyethylene glycol (PEG-CHO) waspurchased from NOF Co., Japan. Acetic acid, acetaldehyde,dimethylsulfoxide-d₆, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), Xanthine,Xanthine oxidase (XO), nitroblue tetrazolium (NBT),2-amino-2-hydroxymethyl-1,3-propanediol (Tris) and polyethylene glycol(PEG) 8000 were purchased from Sigma-Aldrich. Urokinase (uPA) andSPECTROZYME™ UK were purchased from American Diagnostica Inc. Otherreagents and solvents are commercially available and used as received.

Synthesis of OEGCG, PEG-EGCG or PEG-OEGCG: Oligomeric EGCG (OEGCG) andpoly(ethylene glycol) conjugates with EGCG (PEG-EGCG) or OEGCG(PEG-OEGCG) were synthesized as described above. For OEGCG synthesis,EGCG was dissolved in a mixture of acetic acid/water/DMSO. The reactionwas started by addition of acetaldehyde and performed at 20° C. (pH from1 to 5) under a nitrogen atmosphere for 24 hr. The resulting productswere dialyzed (molecular weight cutoff: 1×10³) against 1000 times thevolume of methanol at room temperature for two days, and then theremaining solution was lyophilized to give OEGCG. For conjugatessynthesis, PEG-CHOs and EGCG or OEGCG were separately dissolved in amixture of acetic acid/water/DMSO. The reaction was started by dropwiseaddition of PEG-CHO solution and performed at 20° C. (pH from 1 to 5)under a nitrogen atmosphere for 24 hr. The resulting products weredialyzed in a same way described above (molecular weight cutoff:3.5×10³). The PEG-EGCG and conjugate was obtained by lyophilization ofdialyzed remaining solution. The PEG-OEGCG conjugate was precipitated bycentrifugation (rpm=3.5×10⁴) before dialysis (molecular weight cutoff:5000) and then lyophilized. The molecular weight was estimated by sizeexclusion chromatography (Waters 2690 equipped with RI-2410 detector,polystyrene standard) with Waters Styragel HR4E/HR5E columns using THFas an eluent at a flow rate of 1 ml/min at 40° C., after actetylation.¹H and ¹³C NMR were recorded on a Bruker 400-MHz nuclear magneticresonance (NMR) spectrometer.

Diphenyl-picryl-hydrazyl scavenging activity: Different amounts of asample were mixed with the chemically stable free radical1,1-diphenyl-2-picryl-hydrazyl (DPPH) solution and absorbance at 519 nmwas continuously recorded for 30 min at 25° C. using a UV-visiblespectrophotometer (JASCO V-510 UV/VIS/NIR spectrometer, Japan). Allanalyses were run in triplicate and the results were averaged.

Superoxide anion scavenging activity: Superoxide anion was generatedusing xanthine and xanthine oxidase (XO), and measured by the nitrobluetetrazolium (NBT) reduction method. A test sample was mixed in a buffersolution (pH 7.0) containing xanthine and NBT at 25° C. Measurementbegan with the addition of XO. Production of superoxide anion wasfollowed spectrophotometrically at 560 nm for 10 min at 25° C. using aUV-visible spectrophotometer. All analyses were run in triplicate andthe results were averaged. Superoxide scavenging activity was calculatedaccording to the following formula:

${{Superoxide}\mspace{14mu}{scavenging}\mspace{14mu}{activity}\mspace{11mu}(\%)} = {\frac{{absorbance}_{control} - {absorbance}_{sample}}{{absorbance}_{control}} \times 100}$

Xanthine oxidase inhibitory activity: The activity of XO was measuredspectrophotometrically by monitoring the formation of uric acid at 295nm for 30 min using a UV-visible spectrometer. The assay was carried outunder the same conditions as the superoxide anion assay, and thepercentage activity was calculated.

uPA inhibitory activity: Various amounts of a sample were mixed with uPAin a buffer solution, and incubated for 15 min at 37° C. The mixturesolution was added with SPECTROZYME™ and absorbance at 405 nm wasrecorded for 10 min using a microplate reader.

Results: Oligomerized (−)-epigallocatechin gallate (OEGCG) andconjugates of poly(ethylene glycol) with EGCG (PEG-EGCG) or the oligomer(PEG-OEGCG) were synthesized by the aldehyde-mediated condensationdescribed above. The molecular weights estimated by size exclusionchromatography after acetylation, were Mw=4000, Mw/Mn=1.2; Mw=7900,Mw/Mn=1.2; and Mw=10100, Mw/Mn=1.1 for OEGCG, PEG-EGCG, and PEG-OEGCG,respectively.

Diphenyl-picryl-hydrazyl scavenging activity and Superoxide anionscavenging activity: The 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH)assay, which measures hydrogen atom donating activity, provides anevaluation of antioxidant activity due to free radical scavenging. DPPH,a purple-coloured stable free radical, is reduced into theyellow-coloured diphenylpicry hydrazine, as the radical is scavenged byantioxidants through donation of hydrogen. The compound capable of DPPHscavenging shows decreased absorbance at 519 nm as an indication of freeradical scavenging activity. Addition of sample solutions showedsignificantly decreased absorbance maxima at 519 nm in all cases ofOEGCG, PEG-EGCG and PEG-OEGCG (FIG. 6). The DPPH scavenging activity ofsamples was expressed by IC₅₀ (the concentration needed to scavenge DPPHby 50%), as shown in Table 4. The concentration-dependent free radicalscavenging activities of OEGCG, PEG-EGCG and PEG-OEGCG were amplified,compared to the IC₅₀ observed for intact EGCG. These activities werealso much higher than those of commercial antioxidants, vitamin C anddibutylhydroxytoluen (BHT).

A mixture of xanthine and XO generates superoxide anion, which reducesnitroblue tetrazolium (NBT) to give the blue chromogen formazan andincreases UV absorbance at 560 nm. Compounds capable of scavengingsuperoxide anion, such as superoxide dismutase (SOD), inhibit NBTreduction. We found amplified concentration-dependent SOD-like activitythan that observed for intact EGCG with lower IC₅₀ (the concentrationneeded to scavenge superoxide anion by 50%) in the case of PEG-EGCG,PEG-OEGCG and OEGCG, indicating that these compounds are more potentscavengers against superoxide anion than unmodified EGCG. Sincecompounds capable of scavenging superoxide anion can also affect NBTreduction, samples were investigated for their effects on theseprocesses. A control experiment revealed that the samples did notdirectly reduce NBT in the range of concentrations tested. Evaluation ofscavenging activity against DPPH and superoxide anion provided directevidence of the free radical scavenging potential of those compounds.The results of the DPPH and superoxide anion assays indicated that theantioxidant activity was amplified on an EGCG unit-basis by theoligomerization and/or PEG conjugation of EGCG. These results imply thata single constituent EGCG unit within any of the oligomers andconjugates (OEGCG, PEG-EGCG or PEG-OEGCG) has a more potent scavengingactivity than that of one EGCG unit alone in non-modified form.

Xanthine oxidase inhibitory activity: XO is not only an importantbiological source of reactive oxygen species but also the enzymeresponsible for the formation of uric acid associated with gout leadingto painful inflammation in the joints (McCord J. M. and Fridovich I. J.Biol. Chem. 1968, 243, 5753; Chiang H. C., Lo Y. J. and Lu F. J. J.Enzyme Inhibition 1994, 8, 61). FIG. 7 shows XO inhibitory activityassessed by evaluating uric acid formation from XO. All of OEGCG,PEG-EGCG, and PEG-OEGCG exhibited higher inhibition activities than thatof allopurinol, a frequently used commercial inhibitor for gouttreatment (Feher M. D., et al. Rhermatology 42, 321 (2003)), in aconcentration dependent manner. In contrast, EGCG showed lowerinhibition activity, namely less than about 5% inhibition over the rangeof concentrations tested. The inhibition activities measured using 10 μMof samples were 100, 89.3, 30.7, 22.6, and 1.2% for PEG-OEGCG, OEGCG,PEG-EGCG, and allopurinol, and EGCG, respectively. Since compoundscapable of inhibiting XO can also positively affect the activity toscavenge superoxide radicals, the XO inhibitory activity might partlycontribute to the results showed in Table 4. However, the XO inhibitoryactivity was lower than superoxide radical scavenging activity in arange of tested concentrations. Therefore, the greater inhibition effectof OEGCG and those conjugates on the superoxide anion scavenging appearsto result predominantly from superoxide radical scavenging rather thanfrom XO inhibition. These results demonstrate that the EGCG oligomersand PEG conjugates possess a higher potential for both superoxide anionscavenging and XO inhibition, as compared with unmodified EGCG.

uPA inhibitory activity: Human cancers need proteolytic enzymes toinvade cells and form metastases. One of these enzyme is urokinase(uPA). Inhibition of uPA can decrease tumor size or even cause completeremission of cancers in mice. The known uPA inhibitors are unlikely tobe used in anticancer therapy because of their weak inhibitory activityor high toxicity. EGCG was demonstrated to bind to uPA, blocking His 57and Ser 195 of the uPA catalytic triad and extending towards Arg 35 froma positively charged loop of uPA. Such localization of EGCG wouldinterfere with the ability of uPA to recognized its substrates andinhibit enzyme activity. EGCG showed very low uPA inhibition activityover a range of tested concentrations (FIG. 8). However, OEGCG, PEG-EGCGand PEG-OEGCG showed higher inhibition activities in an EGCG-unitconcentration-dependent manner.

Example 3 Micellar Nanocomplex of OEGCG and PEG-EGCG

Materials: (−)-Epigallocatechin gallate (EGCG) was purchased from KURITALTD., Japan. Aldehyde-terminated polyethylene glycol (PEG-CHO) waspurchased from NOF Co., Japan. Acetic acid, acetaldehyde, bovine serumalbumin (BSA), fluorescein isothiocyanate-bovine albumin (FITC-BA),PURPALD™ and vanillin were purchased from Sigma-Aldrich. Other reagentsand solvents are commercially available and used as received.

Synthesis of OEGCG, PEGCG and POEGCG: Oligomeric EGCG (OEGCG) andpoly(ethylene glycol) conjugates with EGCG (PEG-EGCG) were synthesizedas above. For OEGCG synthesis, EGCG was dissolved in a mixture of aceticacid/water/DMSO. The reaction was started by addition of acetaldehydeand performed at 20° C. (pH from 1 to 5) under a nitrogen atmosphere for24 hr. The resulting products were dialyzed (molecular weight cutoff:1×10³) against 1000 times the volume of methanol at room temperature fortwo days, and then the remaining solution was lyophilized to give OEGCG.For conjugates synthesis, PEG-CHOs and EGCG were separately dissolved ina mixture of acetic acid/water/DMSO. The reaction was started bydropwise addition of PEG-CHO solution and performed at 20° C. (pH from 1to 5) under a nitrogen atmosphere for 24 hr. The resulting products weredialyzed in a same way described above (molecular weight cutoff:3.5×10³). The PEG-EGCG and conjugate was obtained by lyophilization ofdialyzed remaining solution. The molecular weight was estimated by sizeexclusion chromatography (Waters 2690 equipped with RI-2410 detector,polystyrene standard) with Waters Styragel HR4E/HR5E columns using THFas an eluent at a flow rate of 1 ml/min at 40° C., after actetylation(Mw=4000; Mw/Mn=1.2 and Mw=7900; Mw/Mn=1.2 for OEGCG and PEG-EGCG,respectively).

Interaction of oligomeric (−)-epigallocatechin gallate with protein orDNA: 10 μM of OEGCG stock solutions in DMSO or methanol with variousfinal concentrations (0-0.14 mg/ml) were added to 2 ml of BSA solutionin PBS with various concentrations (0-100 mg/ml). Complexes of OEGCG andproteins were formed immediately after mixing by spontaneousself-assembly. The size of complexes was measured at the indicated timesfor 2 days using particle analyzer (ZetaPALS, BROOKHAVEN INSTRUMENTSCo.). Formation of DNA complex with OEGCG was observed in a same way.ζ-potential of the sample solutions was measured at 25° C. using zetapotential analyzer (ZetaPALS, BROOKHAVEN INSTRUMENTS Co.). Eachmeasurement was run in triplicate.

Micellar nanocomplex carrier formation and characterization: 50 μl ofPEG-EGCG solution in DMSO prepared with various concentrations was addedto OEGCG/BSA complex solutions to form the micellar nanocomplex (MNC)carrier. The MNC solution was ultrafiltered three times usingultrafiltration membrane (molecular cut off=200000) to remove an excessof flavonoic compounds and protein which not participated in MNC. Sizeand ζ-potential of MNC were measured at 25° C. using particle analyzerand zeta potential analyzer, respectively. Phenolic content of MNC wasassessed by vanillin-HCl assay. 100 μl of a sample was added to 1 ml of4% vanillin in methanol and the mixture was shaken vigorously. 0.5 ml ofconcentrated HCl was added then, and the mixture was immediately shakenagain. The absorptivity was read at 500 nm after incubating the mixtureat room temperature for 20 min. The phenolic content was determinedusing EGCG standard curves measured in the same manner; each measurementwas run in triplicate. To determine the amount of protein loaded in aMNC carrier, FITC-BA loaded MNC was fabricated in 10 mM Tris (pH 7.0) ina same manner described above and the fluorescence intensity wasmeasured using spectrofluorometer. Wavelengths of excitation andemission were set at 491 nm and 519 nm, respectively. The loadingefficiency of protein was determined by dividing the mass of the loadedprotein by the initial mass of protein in feed. The amount of proteinloaded was expressed as a percentage determined by dividing the mass ofthe loaded protein by the mass of lyophilized MNC. The morphology of MNCwas observed at 200 kV using a transmission electron microscope (TEM)(FEI Tecnai G2 F20 S-Twin). 100 μl of MNC solution stained with 0.001mg/ml of phosphotungstic acid was fixed on a copper grid coated withcarbon film and dried at room temperature for overnight.

Results: In this experiment, complex formation of oligomeric EGCG(OEGCG) with BSA was characterized in terms of the complex size (FIG.9). When OEGCG was added to BSA solution, the size of particles in themixture immediately increased due to complex formation. The size of thecomplex increased with increasing OEGCG concentration at a constant BSAconcentration, while no increase in the size was observed with EGCGaddition in the range of concentrations tested. As the BSA concentrationwas varied, the complex increased in size up to a maximum size, afterwhich point the complex size decreased with increasing BSAconcentration. These results imply that the complex forms as the proteinmolecules are bound by OEGCG molecules (Baxter N. J., et al.Biochemistry, 36, 5566-5577 (1997); Siebert K. J., et al. J. Agric. FoodChem. 44, 80-85 (1996)). Therefore, when the BSA concentration is lessthan a critical amount, OEGCG is present in excess allowing for largerparticles to form. When the protein concentration is high, complexformation is limited to a smaller size as a resulting of fewer OEGCGmolecules available to form a bridge between multiple protein molecules.The complexes were observed to be stable over 2 days.

PEG-EGCG was successively added to the OEGCG/BSA complex solution withincreasing concentrations and the resulting complex size was observed(FIG. 10). Two combinations with different concentrations of OEGCG andBSA were chosen initially to obtain the OEGCG/BSA complex with adiameter of around 30 nm. The size rapidly increased above a certainPEG-EGCG concentration and stopped on around 80 nm, indicating themicellar nanocomplex (MNC) formation by PEG-EGCG assembled surroundingOEGCG/protein complexes. For lower concentrations of BSA, the PEG-EGCGamount needed to form the micelle was relatively smaller, compared tothat needed for higher concentrations of BSA.

ζ-potential measurement the complexes also demonstrated the MNCformation (FIG. 11). Surface charge of OEGCG/BSA complexes showed morenegative charge than either BSA or OEGCG alone. However, the complex hadthe same surface charge as that of PEG alone, after addition of PEG-EGCGto the OEGCG/BSA complexes, indicating that the micellar structure issurrounded by PEG chains.

In the absence of OEGCG, PEG-EGCG still formed micellar complexes withBSA alone by self-assembly between EGCG moiety of PEG-EGCG and BSA (FIG.12). Also, the hydrophobic interaction of EGCG moiety was able to driveself-assembly between PEG-EGCGs themselves and formed micelles in anaqueous solution. However, both of the assembly in the absence of OEGCGwere not stable enough and showed serious reduction of size when proteinwas further added, indicating micelle dissociation. In contrast, thecomplex formed by PEG-EGCG and OEGCG/BSA did not show a change in sizeupon further addition of protein, likely due to OEGCG stabilization ofthe nanocomplex structure by strong hydrophobic and hydrogen bondinginteraction.

To assess the amount of protein loaded in the nanocomplex, a FITC-BAloaded micelle was fabricated and measured. The protein amount loadedwas 39.3% and the loading efficiency was 60.9%. In addition, theflavonoid amount loaded together was determined using the vanillin-HClmethod. When the vanillin assay was used for determination of EGCG unitin OEGCG, PEG-EGCG and PEG-OEGCG, the result was quite reproducible andgave nearly same amount as the amount calculated based on theirmolecular weight. Vanillin-HCl assay revealed 58.5% of flavonoid loadingamount with 7.3% loading efficiency.

Light scattering analysis of the nanocomplex showed a monodispersedparticle size around 80 nm (FIG. 13B). TEM image showed a sphericalcompact shape of the nanocomplex showing good consistency with the sizeobserved by light scattering (FIG. 13A).

FIG. 14 indicates that OEGCG forms complexes with DNA as well. Thecomplex size measured by light scattering increased with increase in theconcentration of EGCG units of OEGCG. Unmodified EGCG was not observedto form complexes with DNA in a range of concentration tested.

Example 4 PEG-OEGCG Micellar Nanocomplex Formation

Materials: (−)-Epigallocatechin gallate (EGCG) was purchased from KURITALTD., Japan. Aldehyde-terminated polyethylene glycol (PEG-CHO) waspurchased from NOF Co., Japan. Acetic acid, acetaldehyde, bovine serumalbumin (BSA) were purchased from Sigma-Aldrich. Other reagents andsolvents are commercially available and used as received.

Synthesis of OEGCG, PEG-EGCG and PEG-OEGCG: Oligomeric EGCG (OEGCG) andpoly(ethylene glycol) conjugates with EGCG (PEG-EGCG) were synthesizedas above. For OEGCG synthesis, EGCG was dissolved in a mixture of aceticacid/water/DMSO. The reaction was started by addition of acetaldehydeand performed at 20° C. (pH from 1 to 5) under a nitrogen atmosphere for24 hr. The resulting products were dialyzed (molecular weight cutoff:1×10³) against 1000 times the volume of methanol at room temperature fortwo days, and then the remaining solution was lyophilized to give OEGCG.For conjugates synthesis, PEG-CHOs and EGCG or OEGCG were separatelydissolved in a mixture of acetic acid/water/DMSO. The reaction wasstarted by dropwise addition of PEG-CHO solution and performed at 20° C.(pH from 1 to 5) under a nitrogen atmosphere for 24 hr. The resultingproducts were dialyzed in a same way described above (molecular weightcutoff: 3.5×10³). The PEG-EGCG and conjugate was obtained bylyophilization of dialyzed remaining solution. The PEG-OEGCG conjugatewas precipitated by centrifugation (rpm=3.5×10⁴) before dialysis(molecular weight cutoff: 5000) and then lyophilized. The molecularweight was estimated by size exclusion chromatography (Waters 2690equipped with RI-2410 detector, polystyrene standard) with WatersStyragel HR4E/HR5E columns using THF as an eluent at a flow rate of 1ml/min at 40° C., after acetylation (Mw=4000, Mw/Mn=1.2; Mw=7900,Mw/Mn=1.2; and Mw=10100, Mw/Mn=1.1 for OEGCG, PEG-EGCG, and PEG-OEGCG,respectively).

Micellar nanocomplex carrier formation: 50 μl of PEG-OEGCG solution inDMSO prepared at various concentrations was added to BSA, EGCG, OEGCGand OEGCG-BSA complex solutions to form the micellar nanocomplex (MNC)carrier. The MNC solution was ultrafiltered three times usingultrafiltration membrane (molecular cut off=200000, ADVANTEC) to removean excess of uncomplexed flavonoic compounds and protein. The size ofMNC was measured at 25° C. using a particle analyzer (ZetaPALS,BROOKHAVEN INSTRUMENTS Co.).

Results: When PEG-EGCG was added to the OEGCG-protein complex formed inadvance, PEG-EGCG spontaneously assembled surrounding the complex andformed micellar complex (MNC) with the complex size around 100 nm.Interestingly, if PEG-EGCG was added directly to OEGCG before complexformation with protein, an insoluble haze-like complex with a sizearound 500 nm was formed (FIG. 15). This may be due to a strong complexformation of OEGCG with the PEG chain. A similar phenomenon was observedupon addition of OEGCG and unmodified PEG, indicating a stronginteraction exists between the OEGCG and PEG chains.

When PEG-OEGCG was added to protein, a large complex formed with acomplex size of above 800 nm (FIG. 16). This complex may be induced byintra- and intermolecular complexation between the PEG segment and theOEGCG segment in the conjugate molecule as well as between theconjugates and protein. Unlike in the PEG-EGCG system, addition ofPEG-OEGCG to an OEGCG-protein complex formed in advance also resulted inlarge complexes, even though the size decreased somewhat upon additionof the PEG-OEGCG. These huge complexes were stable against physicalcrushing energy like ultrasonication.

However, the strong complexation of PEG-OEGCG was significantly affectedby pH and ionic strength of the medium. FIG. 17 shows the reversiblesize changes of PEG-OEGCG complexes as pH is varied in the direction ofthe arrows. Moreover, in distilled water, PEG-OEGCG formed solublecomplex with protein, EGCG, OEGCG, and OEGCG-protein complexes, giving asize of around 100 nm (FIG. 18). The complexes once formed in distilledwater did not showed size increase again, even when they were placedback in PBS, possible because the OEGCG segments were protected insidethe core of the nanocomplex. The strong interaction of OEGCG with PEGmay be attributable to the increase in hydrophobicity and hydrogenbonding of these compounds in acidic and salt-containing solutions.

Example 5 Injectable Biodegradable Hydrogels for Drug Delivery andTissue Engineering

Synthesis of hyaluronic acid-aminoacetylaldehyde diethylacetal (HA-ADD)conjugate: The HA-AAD conjugate ((1) in FIG. 19) was synthesized byfollowing the general protocol as follows. Hyaluronic acid (HA) (1 g,2.5 mmol) was dissolved in 100 ml of distilled water. To this solutionaminoacethlaldehyde dietylacetal (1.2 g, 9 mmol) was added. The pH ofthe reaction mixture was adjusted to 4.7 by the addition of 0.1M HCl.N-Hydroxysuccinimide (0.34 g, 3.0 mmol) and1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC)(0.575 g, 3.0 mmol) were added to the solution. After mixing, the pH ofthe reaction was maintained at 4.7. The solution was kept at roomtemperature for 24 h under gentle stirring. The mixture was subjected topurification by dialysis (molecular weight cut off=1000).

Synthesis of hyaluronic acid-epigallocatechin gallate (HA-EGCG)conjugate: HA-EGCG conjugate was synthesized as follows. 1 g of HA-AADconjugate (1) was dissolved in 60 ml of distilled water. The pH of thesolution was adjusted to 1 by addition of HCl. 5 ml of EGCG solutiondissolved in DMSO (0.2 g/ml) was added. The solution was kept at roomtemperature under nitrogen atmosphere for 24 h with gentle stirring. Themixture was subjected to purification by dialysis (molecular weight cutoff=1000), to yield the HA-EGCG conjugate as shown in FIG. 20.

Hydrogel synthesis and BSA release from the hydrogel: Slab-shapedhydrogels were prepared by injecting a solution mixture of HA-Tyr,HA-Tyr-EGCG containing FITC labeled bovine serum albumin (BSA),horseradish peroxidase (HRP) and H₂O₂ between two glass plates separatedby spacers. After the reaction was complete, the resulting hydrogelswere placed in 50 ml of PBS and examined for BSA release from thehydrogel by measuring the fluorescence intensity of FITC-BSA.

Hydrogels containing EGCG or catechin showed less BSA released comparedto that of HA-Tyr with no catechin-based flavonoid (FIG. 22A and FIG.22B, respectively). This may be due to hydrophobic interactions suchπ-πstacking between proline side-chains in BSA and the EGCG or catechinmoiety in the conjugates. Thus, protein release from the hydrogelscontaining catechin-based flavonoids may be slower, and would havelonger half-life in the body. The hydrogels may also be prepared usingthe HA-EGCG conjugate described above (or another HA-catechin-basedflavonoid conjugate such as HA-catechin) without any tiramine content.In FIG. 22, the hydrogels are composed of varying wt % of catechin-basedflavonoid. For example, HA-Tyr-EGCG40 comprises 60 wt % of HA-Tyr and 40wt % of HA-Tyr-EGCG.

Xanthine oxidase inhibition and superoxide scavenging activity ofHA-EGCG conjugates: These experiments were performed as described above.The results are shown in FIG. 23A and FIG. 23B. In these figures, theratio of conjugated catechin-based flavonoid to the repeating unit of HAis shown in the name of each conjugate. For example, HA-6.8-EGCG meansthat the conjugation degree of EGCG to the repeating unit of HA is 6.8%.

Urokinase inhibition of HA-EGCG conjugates: These experiments wereperformed as described above. The results are shown in FIG. 24.

Example 6 Reversible Activities of Protein andOligo-epigallocatechin-3-gallate (OEGCG) upon Complexation andDissociation

Materials and Methods

Polymer Synthesis and Characterization: To synthesize OEGCG, EGCG(KURITA LTD., Japan) (1 g) was dissolved in a mixture of aceticacid/water/DMSO. The reaction was started by addition of acetaldehyde(7.2 ml) and performed at 20° C. (pH from 1 to 5) under a nitrogenatmosphere for 48 h. The resulting products were dialyzed (molecularweight cutoff=2000) against 1000 times the volume of DMSO graduallyreplacing by distilled water. The remaining solution was lyophilized togive OEGCG. To synthesize PEG-EGCG aldehyde-terminated PEG (PEG-CHO, Mw5000, NOF Co., Japan) (0.35 g) and EGCG (0.65 g) were separatelydissolved in a mixture of acetic acid/water/DMSO. The reaction wasstarted by dropwise addition of PEG-CHO solution and performed at 20° C.(pH from 1 to 5) under a nitrogen atmosphere for 48 h. The resultingproducts were dialyzed (molecular weight cutoff=3500) in a same waydescribed above. The remaining solution was lyophilized to givePEG-EGCG.

Complex characterization: The size and polydispersity of complexes wereevaluated by dynamic light scattering measurements using a 90Plusparticle sizer (Brookhaven instruments Co.). ζ-potential of the samplesolutions was measured at 25° C. using zeta potential analyzer(ZetaPALS, Brookhaven instruments Co.). The morphology of the complexeswas observed at 200 kV using a transmission electron microscope (TEM)(FE1 Tecnai G² F20 S-Twin).

Activity Assessment: Xanthine Oxidase (from buttermilk, Wako Chemical,XO): XO activity was measured by determining uric acid production at 295nm in a UV-Vis spectrophotometer (Hitachi, Japan). The solutioncontained of protein (50 μg/ml) and OEGCG (50 μg/ml)) in a 0.1Mphosphate buffer with or without Triton X-100 (0.1%). Each measurementwas run in triplicate.

α-Amylase (from Apergillus oryzae, Fluka): amylase activity was assayedwith an activity kit from Molecular Probes (E-11954) using afluorescence spectrophotometer (Hitachi, Japan) with excitationwavelength at 505 nm and emission wavelength at 512 nm. The solutioncontained protein (2.5 μg/ml) and OEGCG (2.5 μg/ml) in a 0.1M phosphatebuffer with or without Triton X-100 (0.1%). Each measurement was run intriplicate.

Lysozyme (from egg white, Sigma Chemical): activity of lysozyme wasdetermined spectrophotometrically at 450 nm by the decrease in turbiditydue to the cleavage of glucosidic linkages of micrococcus lysodeikticususing a UV-Vis spectrophotometer (Hitachi, Japan). The solution mixturecontained protein (2.5 μg/ml) and OEGCG (2.5 μg/ml) with or withoutTriton X-100 (0.1%). Each measurement was run in triplicate.

Radical scavenging activity: ABTS^(.+) was prepared by mixing a 7 mMABTS stock solution with 2.45 mM potassium persulfate ((1/1, v/v). Thedecolorization reaction was initiated by adding ABTS^(.+) to samples ina phosphate buffer and immediately measured at 734 nm using a UV-Visspectrophotometer (Hitachi, Japan).

Results: To investigate the interaction mode of the complexation, TritonX-100, Tween 20, sodium dodecyl sulphate (SDS) and urea were added tothe complex (FIG. 25). Complexes were efficiently dissociated by TritonX-100, Tween 20 and SDS due to hydrophobic competition. Relatively highmolecular weight non-ionic detergents, Triton X-100 and Tween 20 weremore effective in dissociating the complexes than SDS. However, urea,which has the ability to participate in the formation of strong hydrogenbonds and is not intrinsically hydrophobic, was ineffective inaccomplishing dissociation of the complexes in the range ofconcentrations tested. This result shows that the dominant mode ofinteraction between OEGCG and protein might be hydrophobic interactionrather than hydrogen bonding.

The polyphenols are renowned for binding to preferred sites and regionson the protein where its aromatic, proline and histidine residues aremost readily accommodated by the development of hydrophobicinteractions. The association is firmly reinforced by the hydrogen bondsbetween phenolic hydroxyl groups of polyphenols and polar groups such asthe carbonyl group in the vicinity of the binding site of the polyphenolto the peptide linkages of protein surface. OEGCG may bind to proteinsin a multidentate fashion at more than one point and cross-linkdifferent protein molecules.

FIG. 26 demonstrates that activities of various proteins were restrainedby complexation with OEGCG, but the activities were fully restored whenthe complexes were dissociated by Triton X-100, implying theconformation change of protein affecting its activity was reversibleupon the dissociation.

On the other hand, the water soluble radical scavenging activity ofOEGCG was investigated as one of representative indexes of itsbioactivities (FIG. 27). Similar phenomena were observed, namely, theradical scavenging activity of OEGCG suppressed by complexation withproteins was restored upon dissociation. This implies that complexationis able to preserve the activities of both protein and OEGCG in adormant state. This property is particularly useful and advantageous inlight of the fact that the micelles need to travel through manydegrading barriers from point of administration to the intended target(e.g., tumor) sites. As well, the activities of protein and OEGCG areconsiderably restored upon the dissociation of the micellar complexes.

Example 7 Inhibition and Restoration of Protein Activity withOligo-epigallocatechin-3-gallate (OEGCG)

Materials and Methods

Enzyme Activity Assays: Xanthine Oxidase from buttermilk, Wako Chemical.XO activity was measured by determining uric acid production in a UV-visspectrophotometer at 295 nm. The increase in absorbance due to theconversion of xanthine to uric acid was followed for 3 minutes. Thesolution contained 50 μg/mL of protein and DMSO with or without OEGCGsamples in a 0.1 M phosphate buffer. To determine the dose dependentresponse of protein to the test compound, protein to OEGCG ratios werevaried from 0.05-5. 164 μM xanthine was added and mixed for 1 minute tostart the reaction.

α-Amylase from Apergillus oryzae, Fluka. Amylase activity was assayedwith an activity kit from Molecular Probes (E-11954) using afluorescence spectrophotometer with excitation wavelength at 505 nm andemission wavelength at 512 nm. The solution contained 2.5 μg/mL ofprotein as well as DMSO with and without OEGCG samples in a 0.1 Mphosphate buffer. The OEGCG concentrations were varied from μM. To startthe reaction, 2.5 μg/mL of fluorescent starch from the Molecular Probeskit was added to the solution and mixed for 30 s. Fluorescence wasmonitored over a period of 3 minutes.

Catalase from bovine liver, Sigma Chemical. Catalase activity wasmeasured by the disappearance of H₂0₂, followed at 240 nm in a UV-Visspectrophotometer over a period of 200 s. The solution mixture contained2.5 μg/mL of protein in the presence and absence of OEGCG in DMSO. Thereaction was started by adding 0.507M H₂O₂ and mixing for 25 s.

Lysozyme from egg white, Sigma Chemical. Activity of lysozyme wasdetermined spectrophotometrically at 450 nm by the decrease in turbidityover time due to the cleavage of glucosidic linkages of Micrococcuslysodeikticus. The solution mixture contained 2.5 μg/mL of protein withand without OEGCG. To start the reaction, 0.25 mg/mL of Micrococcuslysodeikticus was added to the solution.

Activity Recovery with Triton X-100: Recovery of protein activity wasmeasured by adding TX-100 to the reaction mixture of the respectiveenzymes after protein complexation with OEGCG. Activity was measuredaccording to the specified method for each protein.

Activity for all proteins is calculated with the following equation:

${\%\mspace{14mu}{Protein}\mspace{14mu}{Activity}} = \frac{\Delta\;{Abs}_{sample}}{\Delta\;{Abs}_{control}}$where ΔAbs_(sample) is the change in absorbance over time with the testcompound and ΔAbs_(control) is the change in absorbance over timewithout the test compound.

Protein inhibition: The ability of OEGCG to inhibit protein activity isshown in FIG. 28. All proteins show a dose dependent decrease inactivity with increasing levels of OEGCG. The error bars obtained in thefigure are the result of at least a triplicate measure of separateexperiments.

OEGCG had the least inhibitory effect on catalase, which was inhibitedonly 30% at the highest OEGCG to protein ratio. Moreover, the increasein inhibitory activity did not increase extensively beyond the 1 OEGCGto protein ratio concentration; the five fold increase in OEGCGconcentration only resulted in a decrease of protein activity of about6%.

OEGCG suppressed total XO activity by 75% at the highest concentration.The dose dependent response shows that above a ratio of 2, additionalOEGCG did not substantially inhibit protein activity; inhibition onlyincreased about 8%.

Similarly, α-amylase showed a decrease in activity of only 20% as aresult of a five fold increase in OEGCG concentration from a ratio of 1to 5, while the initial 70% activity inhibition occurs with smallamounts of additional OEGCG below a ratio of one. The maximum inhibitionof α-amylase is about 94%.

Lysozyme showed the most inhibition by OEGCG. All activity wassuppressed above an OEGCG ratio of 2 and remains suppressed for higherconcentrations. Similar to the other three proteins, the bulk of theinhibition occurs with the initial amounts of OEGCG.

Recovery of protein activity: The nature of the interaction betweenOEGCG and the various proteins was tested by determining the effect ofTX-100. As FIG. 2-4 indicate, the addition of TX-100 restored proteinactivity after complexation with OEGCG for both α-amylase and lysozymeto approximately 100% of the initial protein activity. FIG. 29 showsthat for α-amylase, protein activity is initially dose dependentlyinhibited by OEGCG, as indicated by the fact that the higher OEGCGconcentration results in lower protein activity without TX-100. AsTX-100 is added to the α-amylase-OEGCG complex, activity very rapidlyincreased to nearly its original value for a range of TX-100concentrations. FIG. 30 shows similar results for lysozyme. Completeinhibition of lysozyme is achieved with OEGCG and recover of proteinactivity is also nearly 100%.

The activity of xanthine oxidase also fully recovered when the weightratio of OEGCG to protein was 1 (see Example 6), but the recovery wasless beyond the ratio of 2 (FIG. 31). Catalase did not show obviousrecovery as well as inhibition (data not shown).

Discussion: Protein inhibition by OEGCG is a non-specific interaction asevidenced by the activity suppression of all four proteins. Inhibitionoccurred despite dissimilarities in molecular weight, protein function,and protein charge and the concentration ratio of OEGCG to protein. Inaddition, the inhibition appears to occur in two different regimes. Inthe first regime, activity substantially decreases with additionalOEGCG. At a critical concentration, additional OEGCG does not increaseinhibition; the inhibition effect becomes almost saturated. It would beconsistent with the fact that once all active sites capable of beinginhibited by OEGCG are blocked, increasing the concentration has littleeffect on the overall enzyme inhibition.

Although the general trend is observed for all proteins, the maximumlevel of inhibition that can be achieved and the rate of the dosedependent response to OEGCG is protein specific. Catalase, a largeprotein with four subunits, shows much less inhibition compared to themuch smaller lysozyme. However, size is only one factor and does notentirely explain the different dose dependent responses to OEGCG. Otherfactors may include the extent of the hydrophobic nature of the enzymeas well as the protein conformation and location of the active site.

Activity recovery with TX-100 establishes the type of interactionbetween OEGCG and the protein. As a hydrophobic competitor to theprotein-OEGCG complex, dissociation to achieve activity recoveryindicates that inhibition occurs through hydrophobic interactions.Activity recovery also establishes the fact that the nature of theOEGCG-protein interaction allows the function of the protein to beregained after dissociation.

As can be understood by one skilled in the art, many modifications tothe exemplary embodiments described herein are possible. The inventionis intended to encompass all such modification within its scope, asdefined by the claims.

All documents referred to herein are fully incorporated by reference.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. All technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art of this invention, unlessdefined otherwise.

TABLE 1 Synthesis of oligomeric EGCG EGCG Acetaldehyde Molar ratio TimeYield sample (g) (g) (EGCG:acetaldehyde) Solvent (ml) (d) atmosphere (%)Mw^(c) Mn^(c) Mw/Mn^(c) OE-1 0.2 1.0 1:52 12.5^(a) 1 Air 23 3600 33001.1 (0.04:0.17:0.79) OE-2 1.0 5.2 1:52 62.4^(a) 14 Air 25 3100 2800 1.1(0.04:0.17:0.79) OE-3 1.0 5.2 1:52 62.4^(a) 2 N₂ 78 4000 3100 1.2(0.04:0.17:0.79) OE-4 0.4 1.8 1:52 23.2^(a) 2 N₂ 28 2400 2100 1.1(0.03:0.02:0.95) OE-5 0.5 2.5 1:52 36.2^(b) 2 N₂ 98 5200 4600 1.1(0.18:0.15:0.67) ^(a)a mixture of acetic acid, ethanol and H₂O, volumeratio in parenthesis, ^(b)a mixture of acetic acid, DMSO and H₂O, volumeratio in parenthesis, ^(c)molecular weight was measured by SEC afteracetylation.

TABLE 2 Synthesis of PEG conjugates with EGCG Molar ratio EGCG (PEG-Time sample PEG-CHO (g) (g) CHO:EGCG) Solvent (ml) (d) atmosphere MW^(d)Mn^(d) Mw/Mn^(d) PEG-CHO(I) 7400 6300 1.2 PE-1 0.65 0.18 1:3  12.1^(a)14 Air 7600 6700 1.1 (0.02:0.5:0.48) PE-2 0.65 0.18 1:3  10.2^(a) 2 N₂7800 6500 1.2 (0.03:0.4:0.57) PE-3 0.27 0.5 1:20  9.5^(a) 2 N₂ 7900 68001.2 (0.03:0.4:0.57) PE-4 0.35 0.65 1:20 12.3^(b) 2 N₂ 7800 6600 1.2(0.17:0.22:0.61) PEG-CHO(II) 12200 10200 1.2 PE-5 0.55 0.5 1:20 12.3^(c)2 N₂ 12500 10600 1.2 (0.03:0.13:0.24:0.6) ^(a)a mixture of acetic acid,ethanol and H₂O, volume ratio in parenthesis, ^(b)a mixture of aceticacid, DMSO and H₂O, volume ratio in parenthesis, ^(c)a mixture of aceticacid, ethanol, DMSO and H₂O, volume ratio in parenthesis, ^(d)molecularweight was measured by SEC after acetylation.

TABLE 3 Synthesis of PEG conjugates with oligomeric EGCG Molar ratioPEG-CHO (PEG- Time Temp. Yield sample (g) OEGCG (g) CHO:OEGCG) Solvent(ml) (d) (° C.) atmosphere (%) Mw^(d) Mn^(d) Mw/Mn^(d) PEG-CHO(I) PO-10.19 0.01 (OE-1) 10:1   2.5^(a) 6 20 Air 7 10300 9000 1.1(0.03:0.4:0.57) 18000 17100 1.1 PO-2 0.28  0.1 (OE-1) 1:1  3.9^(a) 14 20Air 42 10100 8800 1.1 (0.02:0.62:0.36) PO-3 0.61  0.3 (OE-2) 1:116.3^(b) 2 20 N₂ 75 10000 9000 1.1 (0.18:0.15:0.67) PEG-CHO(II) PO-4 1.00.21 (OE-3) 1:1 20.8^(c) 1 50 N₂ 64 15900 13200 1.2(0.02:0.09:0.48:0.41) ^(a)a mixture of acetic acid, ethanol and H₂O,volume ratio in parenthesis, ^(b)a mixture of acetic acid, DMSO and H₂O,volume ratio in parenthesis, ^(c)a mixture of acetic acid, ethanol, DMSOand H₂O, volume ratio in parenthesis, ^(d)molecular weight was measuredby SEC after acetylation.

TABLE 4 Free radical scavenging activity Superoxide DPPH scavengingactivity IC₅₀ radical scavenging Sample (μM) activity IC₅₀ (μM) EGCG 7.3± 0.6^(a) 2.9 ± 0.3^(a) OEGCG 4.9 ± 0.5^(a) 1.1 ± 0.2^(a) PEG-EGCG 6.2 ±0.3^(a) 0.9 ± 0.1^(a) PEG-OEGCG 3.8 ± 0.8^(a) 0.4 ± 0.1^(a) Vitamin C44.7 ± 0.4   30.3 ± 0.3   BHT >>250.0 >>250.0 ^(a)EGCG moietyconcentration of samples, n = 3.

1. A conjugate prepared from reaction of a polymer containing a freealdehyde with a flavonoid, the polymer being conjugated at the C6 and/orthe C8 position of the A ring of the flavonoid.
 2. The conjugate ofclaim 1 wherein the flavonoid is a catechin-based flavonoid.
 3. Theconjugate of claim 1 wherein the flavonoid is a catechin-based flavonoidthat is (−)-epicatechin, (−)-epigallocatechin, (+)-catechin,(−)-epicatechin gallate or (−)-epigallocatechin gallate.
 4. Theconjugate of claim 1 wherein the flavonoid is monomeric or wherein theflavonoid is oligomeric.
 5. The conjugate of claim 4 wherein theflavonoid is oligomeric and is oligomerized through: (i)enzyme-catalyzed oxidative coupling; (ii) aldehyde-mediatedoligomerization; or (iii) a carbon-carbon linkage between the C6 or C8position on the A ring of a first monomeric unit to the C6 or C8position on the A ring of a second monomeric unit.
 6. The conjugate ofclaim 5 wherein the flavonoid is oligomerized via a carbon-carbonlinkage between the C6 position on the A ring of the first monomericunit to the C8 position on the A ring of the second monomeric unit orwherein the flavonoid is oligomeric (−)-epigallocatechin gallate (OEGCG)oligomerized through carbon-carbon C6-C6, C6-C8, C8-C6 or C8-C8 linkagesof (−)-epigallocatechin gallate monomers.
 7. The conjugate of claim 1wherein the polymer is aldehyde-terminated poly (ethylene glycol),aldehyde-derivatized hyaluronic acid, hyaluronic acidaminoacetylaldehyde diethylacetal conjugate, aldehyde-derivatizedhyaluronic acid-tyramine, hyaluronic acid aminoacetylaldehydediethylacetal conjugate-tyramine, cyclotriphosphazene corephenoxymethyl(methylhydrazono) dendrimer or thiophosphoryl corephenoxymethyl(methylhydrazono) dendrimer.
 8. The conjugate of claim 7wherein the polymer is aldehyde-terminated poly (ethylene glycol) andthe conjugate is in the form of a micellar nanocomplex.
 9. The conjugateof claim 8 wherein the micellar nanocomplex comprises the conjugate, theconjugate comprising aldehyde-terminated poly (ethylene glycol)conjugated to an oligomeric catechin-based flavonoid.
 10. The conjugateof claim 8 wherein the micellar nanocomplex comprises an inner corecontaining an oligomeric catechin-based flavonoid and an outer shellcontaining the conjugate, the conjugate comprising aldehyde-terminatedpoly (ethylene glycol) conjugated to a monomeric catechin-basedflavonoid.
 11. The conjugate of claim 10 wherein the oligomericcatechin-based flavonoid is oligomerized through: (i) enzyme-catalyzedoxidative coupling; (ii) aldehyde-mediated oligomerization; or (iii) acarbon-carbon linkage between the C6 or C8 position on the A ring of afirst monomeric unit to the C6 or C8 position on the A ring of a secondmonomeric unit.
 12. The conjugate of claim 11 wherein the oligomericcatechin-based flavonoid is oligomerized via a carbon-carbon linkagebetween the C6 position on the A ring of the first monomeric unit to theC8 position on the A ring of the second monomeric unit or wherein theoligomeric catechin-based flavonoid is oligomeric (-)-epigallocatechingallate (OEGCG) oligomerized through carbon-carbon C6-C6, C6-C8, C8-C6or C8-C8 linkages of (−)-epigallocatechin gallate monomers.
 13. Theconjugate of claim 10 wherein the biological activity of the oligomericcatechin-based flavonoid is masked when the oligomeric catechin-basedflavonoid is assembled in the micellar nanocomplex, and wherein thebiological activity of the oligomeric catechin-based flavonoid isunmasked when the oligomeric catechin-based flavonoid is released fromthe micellar nanocomplex.
 14. The conjugate of claim 7 wherein thepolymer is aldehyde-derivatized hyaluronic acid, hyaluronic acidaminoacetylaldehyde diethylacetal conjugate, aldehyde-derivatizedhyaluronic acid-tyramine, hyaluronic acid aminoacetylaldehydediethylacetal conjugate-tyramine or a combination thereof, and thedelivery vehicle is conjugate is in the form of a hydrogel.
 15. Theconjugate of claim 1 further comprising a bioactive agent.
 16. Theconjugate of claim 15 wherein the bioactive agent is a protein, apeptide, a nucleic acid, a small molecule, a drug, an antibody, ahormone, an enzyme, a growth factor, a cytokine, single stranded DNA,double stranded DNA, single stranded RNA, double stranded RNA, a shorthairpin RNA, an siRNA, an antibiotic, a chemotherapeutic agent or anantihypertensive agent.
 17. The conjugate of claim 1, wherein thebiological activity of the bioactive agent is masked when the bioactiveagent is assembled in the micellar nanocomplex, and wherein thebiological activity of the bioactive agent is unmasked when thebioactive agent is released from the micellar nanocomplex.